Caspases are a family of cysteine proteases that cleave their target protein following an Asp residue. Of the eleven published known caspases, caspase-1, and probably caspases-4, -5, and -11 seem primarily involved in the processing of proinflammatory cytokines, whereas others play crucial roles in the initiation and execution of apoptosis or programmed cell death. Caspases share several common features among them that they are synthesized as catalytically inactive zymogens that are generally activated by cleavage after specific internal Asp residues present in interdomain linkers and the ability to cleave their substrates after Asp residues. As a result certain mature active caspases, in particular those that are derived from the long prodomain caspases can process and activate their own and other inactive caspase zymogens. On the basis of primary structure, proapoptotic caspases can be divided into two classes, class I including caspase-2, -8, -9, and -10 that contain a long amino-terminal prodomain, and class II such as caspase-3, -6, and -7 with a short or absent prodomain. In vitro cleavage experiments suggest that class II caspases require activated class I caspases for their proteolytic processing (12).
Another classification of caspases is by the specificity of the cleave site (13). Here Group 1 caspases have the consensus sequence WEHD (SEQ ID NO: 15) (caspases-1, -4, and -5), Group II caspases the consensus sequence DExD (caspases-2, -3 and -7), and Group III caspases the consensus sequence (IVL)ExD (caspases-6, -8 and -9). The caspase-8 optimal site is LETD (SEQ ID NO: 16) (14).
Interleukin 18 (IL-18), initially described as an interferon-γ. (IFN-γ)-inducing factor is a recently characterized cytokine that shares structural features with the IL-1 family of proteins (1–4). IL-18 was initially purified and subsequently cloned from the liver of mice treated with heat-inactivated Propionbacterium acnes (P. acnes) followed by lipopolysaccharide (LPS) (2,5). Cloning of human IL-18 has also been recently described (3).
Like IL-12, IL-18 is produced by activated macrophages such as liver Kupffer cells and other resident macrophages (3). IL-18 is an early inducer of the Th1 response, co-stimulating the production of IFN-γ, as well as TNF-γ, granulocyte-macrophage colony-stimulating factor and IL-2 (6). In addition, it potentiates anti-CD3-induced T-cell proliferation and increases natural killer cell cytotoxicity by augmenting the expression of Fas Ligand (6,7).
Unlike most other cytokines, which exhibit a four-helix bundle structure, IL-18 and IL-1β have an all β-pleated sheet structure (7). Similarly to IL-1β, IL-18 is synthesized as a biologically inactive precursor (proIL-18), lacking a signal peptide (3). The IL-1β and IL-18 precursors are cleaved by caspase 1 (IL-1β-converting enzyme, or ICE), that cleaves after an aspartic acid residue in the P1 position. The resulting mature cytokines are readily released from the cell (8,9). Studies with caspase-1-deficient mice demonstrated the important role of mature IL-18 as an inducer of IFN-γ and Th1 responses. Injection of such mice with P. acnes and LPS resulted in low levels of circulating IFN-γ compared with wild-type mice (8,9). Injection of IL-18 restored the LPS-induced IFN-γ level in the caspase-1-deficient mice (9), further supporting the concept that caspase-1 is involved in the production of active IL-18. Other caspases and particularly those cleaving intracellular proteins associated with apoptosis were at least 100-fold less active than caspase-1 (9). Similar studies with IL-18-deficient mice revealed its role in NK cell activity and cytokine induction (10).
Recombinant IL-1β and mouse IL-18 expressed in E. coli may be refolded to produce a fully active cytokine. Attempts to express the mature form of human IL-18 in E. coli or other hosts did not provide a fully active cytokine. Because of its potential therapeutic uses, e.g. in malignancies, or in any condition in which induction of interferon-γ production is wanted, it is desired to establish an efficient expression system for the production of mature biologically active human IL-18.